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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3' long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.
Mol Cell Biol 1999 Dec
PMID:In vivo activity of murine de novo methyltransferases, Dnmt3a and Dnmt3b. 1056 46

DNA methylation, a covalent modification of the genome, is emerging as an important player in the regulation of gene expression. This review discusses the different components of the DNA methylation machinery responsible for replicating the DNA methylation pattern. Recent data have changed our basic understanding of the DNA methylation machinery. A number of DNA methyltransferases (DNMT) have been identified and a demethylase has recently been reported. Because the DNA methylation pattern is critical for gene expression programs, the cell possesses a number of mechanisms to coordinate DNA replication and methylation. DNMT1 levels are regulated with the cell cycle and are induced upon entry into the S phase of the cell cycle. DNMT1 also regulates expression of cell-cycle proteins by its other regulatory functions and not through its DNA methylation activity. Once the mechanisms that coordinate DNMT1 and the cell cycle are disrupted, DNMT1 exerts an oncogenic activity. Tumor suppressor genes are frequently methylated in cancer but the mechanisms responsible are unclear. Overexpression of DNMT1 is probably not responsible for the aberrant methylation of tumor suppressor genes. Unraveling how the different components of the DNA methylation machinery interact to replicate the DNA methylation pattern, and how they are disrupted in cancer, is critical for understanding the molecular mechanisms of cancer.
Prog Nucleic Acid Res Mol Biol 2001
PMID:Regulation of the DNA methylation machinery and its role in cellular transformation. 1155 Jul 98

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.
Mol Cell Biol 2002 Jan
PMID:Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. 1175 44

Recent studies have shown that cytosine-5 methylation at CpG islands in the regulatory sequence of a gene is one of the key mechanisms of inactivation. The enzymes responsible for CpG methylation are DNA methyltransferase (DNMT) 1, DNMT3a, and DNMT3b, and the enzyme responsible for demethylation is DNA demethylase (MBD2). Studies on methylation-demethylation enzymes are lacking in human prostate cancer. We hypothesize that MBD2 enzyme activity is repressed and that DNMT1 enzyme activity is elevated in human prostate cancer. To test this hypothesis, we analyzed enzyme activities, mRNA, and protein levels of MBD2 and DNMT1, DNMT3a, and DNMT3b in human prostate cancer cell lines and tissues. The enzyme activities of DNMTs and MBD2 were analyzed by biochemical assay. The mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction and by Northern blotting. The protein expression was measured by immunohistochemistry with specific antibodies. The results of these experiments demonstrated that (1) the activity of DNMTs was twofold to threefold higher in cancer cell lines and cancer tissues, as compared with a benign prostate epithelium cell line (BPH-1) and benign prostatic hyperplasia (BPH) tissues; (2) MBD2 activity was lacking in prostate cancer cell lines but present in BPH-1 cells; (3) immunohistochemical analyses exhibited higher expression of DNMT1 in all prostate cancer cell lines and cancer tissues, as compared with BPH-1 cell lines and BPH tissues; (4) MBD2 protein expression was significantly higher in BPH-1 cells and lacking in prostate cancer cell lines and, in BPH tissues, MBD2 protein expression was poorly observed, as compared with no expression in prostate cancer tissues; and (5) mRNA expression for DNMT1 was upregulated in prostate cancer, as compared with BPH-1, and mRNA expression for MBD2 was found to be significantly expressed in all cases. The results of these studies clearly demonstrate that DNMT1 activity is upregulated, whereas MBD2 is repressed at the level of translation in human prostate cancer. These results may demonstrate molecular mechanisms of CpG hypermethylation of various genes in prostate cancer.
Mol Carcinog 2002 Mar
PMID:DNA methyltransferase and demethylase in human prostate cancer. 1187 Aug 82

We have introduced DNA methyltransferase 1 (Dnmt1) mutations into a mouse strain deficient for the Mlh1 protein to study the interaction between DNA mismatch repair deficiency and DNA methylation. Mice harboring hypomorphic Dnmt1 mutations showed diminished RNA expression and DNA hypomethylation but developed normally and were tumor free. When crossed to Mlh1(-/-) homozygosity, they were less likely to develop the intestinal cancers that normally arise in this tumor-predisposed, mismatch repair-deficient background. However, these same mice developed invasive T- and B-cell lymphomas earlier and at a much higher frequency than their Dnmt1 wild-type littermates. Thus, the reduction of Dnmt1 activity has significant but opposing outcomes in the development of two different tumor types. DNA hypomethylation and mismatch repair deficiency interact to exacerbate lymphomagenesis, while hypomethylation protects against intestinal tumors. The increased lymphomagenesis in Dnmt1 hypomorphic, Mlh1(-/-) mice may be due to a combination of several mechanisms, including elevated mutation rates, increased expression of proviral sequences or proto-oncogenes, and/or enhanced genomic instability. We show that CpG island hypermethylation occurs in the normal intestinal mucosa, is increased in intestinal tumors in Mlh1(-/-) mice, and is reduced in the normal mucosa and tumors of Dnmt1 mutant mice, consistent with a role for Dnmt1-mediated CpG island hypermethylation in intestinal tumorigenesis.
Mol Cell Biol 2002 May
PMID:DNA methyltransferase deficiency modifies cancer susceptibility in mice lacking DNA mismatch repair. 1194 Jun 49

A common event in the development of human neoplasia is the loss of growth regulatory tumor suppressor functions. Methylation of 5'-CpG islands of tumor suppressor genes and elevated levels of the DNA-(cytosine-5)-methyltransferase enzymes (DNMT1, 3A and 3B) are also prevalent features of human neoplasia. However, direct evidence that elevated DNMT enzyme levels alter gene expression and influence oncogenesis has been difficult to obtain due to the lack of specific DNMT inhibitors. We have developed potent and selective antisense inhibitors of the known DNA methyltransferases. MG-98, a second-generation DNMT1-specific antisense inhibitor currently in phase II clinical trials, reactivates silenced tumor suppressor genes and inhibits the growth of cancer cells in vitro and in preclinical in vivo models. Here, we will review the discovery and development of MG-98 as a cancer therapeutic.
Curr Opin Mol Ther 2002 Apr
PMID:Selective inhibition of DNA methyltransferase enzymes as a novel strategy for cancer treatment. 1204 34

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.
Mol Cell Biol 2003 Apr
PMID:Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo. 1266 73

Hypermethylation associated silencing of the CpG islands of tumor suppressor genes is a common hallmark of human cancer. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using two molecular screenings for differentially methylated loci [differential methylation hybridization (DMH) and amplification of inter-methylated sites (AIMS)], we found that DKO cells, but not the single DNMT1 or DNMT3b knockouts, have a massive loss of hypermethylated CpG islands that induces the re-activation of the contiguous genes. We have characterized a substantial number of these CpG island associated genes with potentially important roles in tumorigenesis, such as the cadherin member FAT, or the homeobox genes LMX-1 and DUX-4. For other genes whose role in transformation has not been characterized, such as the calcium channel alpha1I or the thromboxane A2 receptor, their re-introduction in DKO cells inhibited colony formation. Thus, our results demonstrate the role of DNMT1 and DNMT3b in CpG island methylation associated silencing and the usefulness of genetic disruption strategies in searching for new hypermethylated loci.
Hum Mol Genet 2003 Sep 01
PMID:Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases. 1291 69

We have examined the metaphase chromosomal localization of 15 proteins that have previously been described as involved in mammalian chromatin modification and/or transcriptional modulation. Immunofluorescence data indicate that all the proteins localize to human and mouse centromeres, a neocentromere, and the active centromere of a dicentric chromosome, with six of these proteins (Sin3A, PCAF, MYST, MBD2, ORC2, P300/CBP) being demonstrated at mammalian centromeres for the first time. Most of these proteins fall into two distinct chromosomal distribution patterns: (a) kinetochore-associated proteins (Sin3A, PCAF, MYST and BAF180), which colocalize with metaphase kinetochores, but not any of the pericentric and other major heterochromatic regions; and (b) heterochromatin-associated proteins (MeCP2, MBD1, MBD2, ATRX, HP1alpha, HDAC1, HDAC2, DNMT1 and DNMT3b), which colocalize with centromeric/pericentric heterochromatin and all other major heterochromatic sites. A heterogeneous third group (c) consists of the origin recognition complex subunit ORC2 and the histone acetyltransferase P300/CBP, which associate generally with kinetochores in humans and centromeric/pericentric heterochromatin in mouse, with some minor differences in localization. These observations indicate an extensive sharing of protein components involved in chromatin modification at gene loci, centromeres and various chromosomal heterochromatic landmarks. The definition of distinct patterns of chromosomal distribution for these proteins provides a useful basis for the further investigation of the broad-ranging roles of these proteins.
Hum Mol Genet 2003 Dec 01
PMID:Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns. 1451 86

During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5' region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2'-deoxycytidine followed by zebularine hindered the remethylation of the p16 5' region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.
Mol Cell Biol 2004 Feb
PMID:Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. 1472 71


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